Cancer is a leading cause of death, second only to heart disease, of both men and women. In the fight against cancer, numerous techniques have been developed and are the subject of current research, directed to understanding the nature and cause of the disease, and to provide techniques for control or cure thereof.
Since most cancers occur in tissues which are steroid target tissues, treatments using steroid therapy have been useful. Some steroids (e.g., progesterone, glucocorticoids) and antisteroids (e.g., the antiestrogen, tamoxifen) inhibit cell division in most cells. The use of pharmacologic doses of estrogens, antiestrogens or reduction of estrogen (by ablative surgery) have also proven to be effective therapies for the control of breast cancer. For example, the nonsteroidal antiestrogen, tamoxifen, competes with estrogens for cytosol estrogen receptors and thus blocks the effects of estrogen in the target tissue. Progestins have been used to treat endometrial cancer and glucocorticoids have been used to treat leukemias.
Despite the above, it has been found clinically that within each class of cancer, certain populations do not respond to steroid or to antisteroid therapy. Much research has been directed to determining why this is so, both to provide a clinical diagnostic tool so that time will not be lost on steroid or antisteroid therapy for those patients having cancers that will not respond, and also to provide insight into the nature of the disease.
A known difference between steroid "target" and "nontarget" tissues is the presence of specific receptor proteins for steroids in target cells which serve as intermediaries in the action of these hormones. The generally accepted mechanism of action of steroids or antisteroid compounds in target cells is 1) entrance of the compound into cells and binding of this compound to specific receptor proteins, 2) activation of the receptor and the translocation and binding of this steroid receptor complex from somewhere in the cytoplasm or nucleus to the nuclear acceptor sites on the chromosomal material, 3) alteration of transcription of a multitude of genes into messenger RNA (mRNA), 4) processing of the mRNA, and 5) translation of these mRNAs into proteins which perform or serve a variety of functions.
Procedures have been developed to assay for the presence of these receptor proteins. In approximately 20% of cancers from steroid target tissues that have been studied, the steroid receptors are very low in concentration or are absent. As expected, these particular cancers do not respond to steroid therapy. Patients with negative receptor assays also do not respond to non-steroidal antisteroids such as tamoxifen.
However, in studies of steroid therapy in receptor-positive cancer patients, only about half of these cancers (approximately 40% of all cancers) respond to steroid therapies. Examples of this are found in tamoxifen treatments of breast cancers and progesterone treatment of endometrial cancers. It has been speculated that the defects in the steroid action pathway are related to steps subsequent to the binding of the steroid to its receptor.
That defective (nonfunctional) receptors occur and may play a role in the nonresponse of many breast cancers to steroid therapy was reported by Leake and co-workers. As used herein with respect to receptors, the term "nonfunctional" indicates receptors incapable of activation and binding to chromosomal material. Such receptors can bind steroids or antisteroids but are otherwise defective with respect to the steroid action pathway. Studies by this group reported that 25 to 30% of 461 estrogen receptor (ER)-positive breast tumors showed no nuclear estrogen receptor (ERN), i.e., the receptors were nonfunctional (Laing et al., Brit. J. Cancer, 43, 59 (1977); Leake et al., The Lancet, 168 (1981)). In these studies, the cytosol estrogen receptor (ERC) was isolated from the tumors and the standard charcoal receptor quantitation assay performed according to the method of S. G. Korenman, J. Clin. Endocrinol. Metal., 28, 127 (1968). The ERN was assayed by incubating the nuclear pellet obtained from homogenized tissue with [.sup.3 H]-estradiol and determining the amount of ERN present. Similar results were obtained in another laboratory using an in vitro system by incubating crude [.sup.3 H]-ERC with isolated nuclei and assaying the salt-extracted radioactivity (Fazekas and MacFarlane, The Lancet, 565 (1982)). In both studies, a 75 to 100% correlation was found between the presence of a nuclear receptor (i.e., presence of a functional receptor) and the tumor response to steroid therapy.
T. Thorsen et al., J. Steroid Biochem., 10, 595 (1979) disclose a non-quantitative assay for the presence of functional nuclear steroid receptors involving the incubation of whole tissue slices with non-saturating amounts of [.sup.3 H] estradiol.
Thus, assays effective to measure functional/nonfunctional steroid receptors allow a more accurate prediction of which patients will or will not respond to steroid or to antisteroid treatment than do assays which simply determine the presence or absence of receptors. Reliable assays of this type will greatly assist the clinician in planning a course of treatment for these patients and would avoid the loss of treatment time due to the use of ineffective therapy. Although the Leake and MacFarlane methods have demonstrated success in discriminating between functional and nonfunctional nuclear receptors, a need exists for improved nuclear binding assays which (a) are able to analyze the functionality of all of the receptors in a cell, (b) do not require the isolation of the nuclear receptor, (c) do not require cell-free incubations, (d) require less tissue and/or (e) can be performed rapidly with readily-available equipment.